The Na,K-ATPase comprises a group of isozymes responsible for maintaining the Na+ and K+ gradients across the plasma membrane of most cells. Isozyme diversity for the Na,K-ATPase results from the association of different molecular forms of the catalytic alpha, alpha1, alpha2, alpha3 and alpha4 and glycosylated beta (beta1,beta2 and beta3) subunits that constitute the enzyme. The various Na,K-ATPase isozymes are expressed in a highly regulated manner, depending on cell type, developmental stage, hormonal stimulation and pathological state of the tissue. In addition, Na,K-ATPase isoforms have enzymatic properties that are unique. The a4 isoform exhibits the most restricted pattern of expression, and it is selectively expressed in rat testes, where it is abundant in germ cells. Its function comprises approximately two thirds of the total Na,K-ATPase activity of spermatozoa, the rest corresponding to the ubiquitously expressed ctl isoform. Functionally, the alpha4 polypeptide has enzymatic properties different from those of the other Na,K-ATPase isoforms. This suggests a physiological role for the isoform, and (4 may be adapted to fulfill the ionic requirements of the male germ cells. It is well known that the Na+ and K+ gradients are key factors for sperm motility. Also, for spermatozoa capacitation and acrosomal reaction, which are required steps for the biogenesis of fertile spermatozoa. At present, the precise role of (4 in these processes remains unknown. The main objective of the present proposal is to elucidate the biological importance of the Na,K-ATPase alpha4 isoform in male germ cell function and its relevance to male fertility. Specifically the aims are: 1) to investigate expression and cell localization of the Na,K-ATPase (4 polypeptide in male germ cells during development, and in Sertoli cells, 2) to study the function of (4 in male germ cells during gametogenesis, epididymal maturation and acrosomal reaction, 3) to study the role of the Na,K-ATPase (4 isoform in sperm physiology, and 4) to determine the enzymatic properties and function of Na,K-ATPase (4 isoform from humans. This study will be important to understand the biological relevance of the testes specific isoform of the Na,K-ATPase in male fertility and contraception.